Ken Sawada

Cognitive reserve, presynaptic proteins and dementia in the elderly

Differences in cognitive reserve may contribute to the wide range of likelihood of dementia in people with similar amounts of age-related neuropathology. The amounts and interactions of presynaptic proteins could be molecular components of cognitive reserve, contributing resistance to the expression of pathology as cognitive impairment. We carried out a prospective study with yearly assessments of N=253 participants without dementia at study entry. Six distinct presynaptic proteins, and the protein–protein interaction between synaptosomal-associated protein 25 (SNAP-25) and syntaxin, were measured in post-mortem brains. We assessed the contributions of Alzheimers disease (AD) pathology, cerebral infarcts and presynaptic proteins to odds of dementia, level of cognitive function and cortical atrophy. Clinical dementia was present in N=97 (38.3%), a pathologic diagnosis of AD in N=142 (56.1%) and cerebral infarcts in N=77 (30.4%). After accounting for AD pathology and infarcts, greater amounts of vesicle-associated membrane protein, complexins I and II and the SNAP-25/syntaxin interaction were associated with lower odds of dementia (odds ratio=0.36–0.68, P<0.001 to P=0.03) and better cognitive function (P<0.001 to P=0.03). Greater cortical atrophy, a putative dementia biomarker, was not associated with AD pathology, but was associated with lower complexin-II (P=0.01) and lower SNAP-25/syntaxin interaction (P<0.001). In conclusion, greater amounts of specific presynaptic proteins and distinct protein–protein interactions may be structural or functional components of cognitive reserve that reduce the risk of dementia with aging.

Abnormalities of presynaptic protein CDCrel‐1 in striatum of rats reared in social isolation: relevance to neural connectivity in schizophrenia

Post‐weaning social isolation‐rearing of rats leads to behavioural and neurochemical sequelae that model aspects of schizophrenia, and it may be useful to test hypotheses related to putative molecular mechanisms of the illness. In humans, the presynaptic protein CDCrel‐1 represents an interesting candidate molecule for the mechanism and aetiology of schizophrenia. CDCrel‐1 modulates dopamine neurotransmission, binds to the SNARE protein syntaxin and maps onto a region of chromosome 22q11 deleted in velo‐cardio–facial and DiGeorge syndromes, which are associated with increased prevalence of schizophrenia. Using the isolation‐rearing model, we measured immunoreactivity of the synaptic proteins CDCrel‐1, synaptophysin and syntaxin. Male, Sprague–Dawley rats were raised in groups or in isolation for 12 weeks from weaning. Synaptic protein immunoreactivities were measured in striatal and hippocampal homogenates, using a sensitive enzyme‐linked immunoadsorbent assay with monoclonal antibodies. Isolation‐rearing produced region‐ and protein‐specific effects. CDCrel‐1 immunoreactivity was significantly lower in the striatum and marginally higher in the hippocampus of isolation‐reared compared with socially reared animals. There were no statistically significant differences in synaptophysin immunoreactivity in either region. Confocal microscopy demonstrated a high degree of colocalization between the two presynaptic proteins. In striatum, a robust relationship between CDCrel‐1 and syntaxin immunoreactivities was observed in socially reared rats, this was lost in the isolation‐reared animals. Altered levels of the septin CDCrel‐1 in isolation‐reared rats may contribute to changes in neuronal connectivity and neurotransmission, and suggest a potential role for CDCrel‐1 in schizophrenia related to chromosome 22q11 deletion syndrome.

Purification and Characterization of a Trypsin-Like Serine Proteinase from Rat Brain Slices that Degrades Laminin and Type IV Collagen and Stimulates Protease-Activated Receptor-2

Abstract: A trypsin‐like serine proteinase was purified from the incubation medium of rat brain slices by gelatin zymography. The purification consisted of ammonium sulfate precipitation, benzamidine‐Sepharose 6B affinity chromatography, and carboxymethyl‐cellulose and gel filtration chromatographies. The gelatinolytic activity, identified at 22 kDa (P22) by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis under nonreducing conditions, was eluted as one active peak throughout the purification, and the final preparation gave a single protein peak on reverse‐phase HPLC. Diisopropyl fluorophosphate, benzamidine, p‐toluenesulfonyl‐L‐lysine chloromethyl ketone, and aprotinin completely inhibited the activity of P22, whereas phenanthroline, p‐toluenesulfonyl‐L‐phenylalanine chloromethyl ketone, and elastinal did not. P22 efficiently digested the extracellular matrix proteins laminin and type IV collagen. P22 produced an increase in intracellular Ca2+ concentration in A172 glioblastoma, which was desensitized through prior stimulation with protease‐activated receptor‐2 agonist peptide SLIGKV, indicating that P22 can stimulate protease‐activated receptor‐2. Rat brain penetration injury induced gelatinolytic activity in the lesioned area whose molecular size was consistent with that of P22. These results indicated that on incubation of rat brain slices, a trypsin‐like serine proteinase was secreted into the medium that was capable of digesting extracellular matrix and stimulating protease‐activated receptor‐2. It is suggested that the gelatinolytic activity induced by brain injury might be that of P22.

Neuronal and vascular localization of histamine N-methyltransferase in the bovine central nervous system.

Histamine N‐methyltransferase (HMT) (EC 2.1.1.8) plays a crucial role in the inactivation of the neurotransmitter histamine in the CNS. However, the localization of HMT remains to be determined. In the present study, we investigated immunohistochemical localization of HMT in the bovine CNS using a polyclonal antibody against bovine HMT. The HMT‐like immunoreactivity was observed mainly in neurons. Strongly immunoreactive neurons were present in the oculomotor nucleus and ruber nucleus in the midbrain, the facial nucleus in the pons, the dorsal vagal nucleus and hypoglossal nucleus in the medulla oblongata and in the anterior horn as well as intermediolateral zone of the spinal cord. Intermediately immunoreactive neurons were present in the piriform cortex and the inferior olivary nucleus. The grey matter of the forebrain regions was diffusely and faintly stained. In the cerebellum and the striatum, the nerve fibres in the white matter were positive. The tuberomammillary nucleus, where histaminergic neurons are present, were weakly positive. The other immunoreactive structures in the CNS were blood vessels. Almost all of the blood vessel walls, irrespective of whether they were arterial or venous, were variably stained. The glial fibrillary acidic protein‐ (GFAP‐) immunoreactive astrocytes were not stained. These findings indicated that histamine released from histaminergic nerve terminals or varicose fibres is methylated mainly in postsynaptic or extrasynaptic neurons rather than in astrocytes. The localization of HMT in the blood vessel wall may mean that blood‐borne histamine and histamine released from mast cells associated with the blood vessels are catabolized in this structure.

The effects of stimulating protease-activated receptor-1 and -2 in A172 human glioblastoma.

SummaryHuman glioblastoma cell line A172 expressed protease-activated receptor-1 and -2 (PAR-1 and PAR-2). We investigated the effects of the stimulation of these receptors by receptor-activating agonist peptides on the Ca2+ signaling, protein kinase C translocation, cell morphology and cell proliferation in A172. Both PAR-1 agonist SFLLRN and PAR-2 agonist SLIGKV induced an increase in [Ca2+]i. The prior treatment of A172 with PAR-2 agonist SLIGKV did not influence the [Ca2+]i response to PAR-1 agonist SFLLRN or thrombin, however, the prior treatment with PAR-1 agonist SFLLRN or thrombin completely abolished the second response to PAR-2 agonist SLIGKV. Treatment with each agonist peptide produced thinner and fewer processes in A172. The PAR-2 agonist inhibited the proliferation of A172 significantly while PAR-1 agonist did not. PKC-α and γ were translocated from cytosol to membrane with either PAR-1 or PAR-2 stimulation, however, ı was specifically translocated with SFLLRN, and λ with SLIGKV, respectively. These results indicated that PAR-1 and PAR-2 stimulation produced a similar [Ca2+]i response and morphological changes in A172 glioblastoma while the effects on the cell proliferation and activation of PKC isozymes were distinct, suggesting that different signal transduction pathways were activated by these receptors. The uni-directional cross desensitization implies a functional linkage between PAR-1 and PAR-2 receptors.

Immunohistochemical localization of histamine N-methyltransferase in guinea pig tissues.

Histamine plays important roles in gastric acid secretion, inflammation, and allergic response. Histamine N-methyltransferase (HMT; EC 2.1.1.8) is crucial to the inactivation of histamine in tissues. In this study we investigated the immunohistochemical localization of this enzyme in guinea pig tissues using a rabbit polyclonal antibody against bovine HMT. The specificity of the antibody for guinea pig HMT was confirmed by Western blotting and the lack of any staining using antiserum preabsorbed with purified HMT. There was strong HMT-like immunoreactivity (HMT-LI) in the epithelial cells in the gastrointestinal tract, especially in the gastric body, duodenum, and jejunum. The columnar epithelium in the gallbladder was also strongly positive. Almost all the myenteric plexus from the stomach to the colon was stained whereas the submucous plexus was not. Other strongly immunoreactive cells included the ciliated cells in the trachea and the transitional epithelium of the bladder. Intermediately immunoreactive cells included islets of Langerhans, epidermal cells of the skin, alveolar cells in the lung, urinary tubules in the kidney, and epithelium of semiferous tubules. HMT-LI was present in specific structures in the guinea pig tissues. The widespread distribution of HMT-LI suggests that histamine has several roles in different tissues.

Interaction of the delta and b subunits contributes to F1 and F0 interaction in the Escherichia coli F1F0-ATPase.

Interactions of the F1F0-ATPase subunits between the cytoplasmic domain of the b subunit (residues 26–156, bcyt) and other membrane peripheral subunits including α, β, γ, δ, ε, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitroreconstitution of ATPase from the purified subunits as well. Only the combination of bcyt fused to the activation domain of the yeast GAL-4, and δ subunit fused to the DNA binding domain resulted in the strong expression of the β-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of bcyt fused to glutathione S-transferase (GST) together with the δ subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-bcyt fusion alone had no such effect, indicating that GST-bcyt was protected by the co-expressed δ subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the δ subunit in the cell. The affinity purified GST-bcyt did not contain significant amounts of δ, suggesting that the interaction of these subunits was relatively weak. Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits. The ATPase activity was reconstituted from the purified bcyttogether with α, β, γ, δ, and ε, or with the same combination except ε. Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex. The result indicated that interaction of b and δ was stabilized by F1 subunits other than ε and also suggested that b-δ interaction was important for F1-F0interaction.

COMBINATION TREATMENT OF HYDROGEN PEROXIDE AND X-RAYS INDUCES APOPTOSIS IN HUMAN PROSTATE CANCER PC-3 CELLS

PURPOSE To study the effect of hydrogen peroxide (H(2)O(2)) on radiation-induced apoptosis in human prostate cancer PC-3 cells. METHODS AND MATERIALS At 4h before the irradiation, PC-3 cells were exposed to 10mM ammonium chloride (NH(4)Cl) concentrations. Subsequently, cells were exposed to 0.1mM H(2)O(2) just before the irradiations, which were administered with 10-MV X-rays at doses of 10 Gy. RESULTS The percentage of apoptotic cells at 48 h after X-irradiation alone, H(2)O(2) alone, and combined X-irradiation and H(2)O(2) was 1.85%, 4.85%, and 28.4%, respectively. With use of combined X-irradiation and H(2)O(2), production of reactive oxygen species (ROS) occurred 4h after the irradiation. This resulted in lysosomal rupturing, mitochondrial fragmentation, and the release of cytochrome c into the cytoplasm from the mitochondria. In contrast, when cells were exposed to NH(4)Cl before the X-irradiation and H(2)O(2) administration, apoptosis was almost completely suppressed, ROS production did not occur, lysosomal rupture and mitochondrial fragmentation were blocked, and cytochrome c was not released. CONCLUSIONS Hydrogen peroxide strongly enhanced lysosome-dependent radiation-induced apoptosis in human prostate cancer PC-3 cells. A combined use of X-rays and H(2)O(2) can also injure the mitochondrial cytoplasmic organelles and lead to the production of ROS that in and of itself might possibly induce apoptosis.

Vulnerability of synaptic plasticity in the complexin II knockout mouse to maternal deprivation stress.

The alterations in brain function and structure seen in schizophrenia are mediated by genetics as well as vulnerability due to environmental factors. Postmortem studies in schizophrenic patients have shown that expression of complexin II, which is involved in neurotransmitter release at central nervous system synapses, is decreased in the brain. We examined the physiological characteristics of complexin II gene-deficient mice subjected to maternal deprivation stress to determine whether psychological stress during the early stage of life affected the development of brain function. We compared the electrophysiological properties of CA1 hippocampal pyramidal neurons and spatial memory in the Morris water maze test in the wild-type mouse and the homozygous mutant. In the non-stressed mouse, no significant differences in transsynaptic responses and synaptic plasticity or spatial memory were seen, suggesting that complexin II does not play a critical role in transmitter release or synaptic plasticity under these conditions. In contrast, under conditions of maternal deprivation stress, the knockout mouse showed a significant decrease in post-tetanic potentiation and LTP induction and a significant impairment in Morris water Maze test compared to the wild-type mouse, suggesting that complexin II plays a significant role in neurotransmitter release and synaptic plasticity under this pathological condition. Taken together, these results show that mice lacking complexin II are vulnerable to maternal deprivation stress, which raises the possibility that the complexin II gene may be a factor in the onset of schizophrenia.

Hippocampal Subfield Volumes in First Episode and Chronic Schizophrenia

Background Reduced hippocampal volume in schizophrenia is a well-replicated finding. New imaging techniques allow delineation of hippocampal subfield volumes. Studies including predominantly chronic patients demonstrate differences between subfields in sensitivity to illness, and in associations with clinical features. We carried out a cross-sectional and longitudinal study of first episode, sub-chronic, and chronic patients, using an imaging strategy that allows for the assessment of multiple hippocampal subfields. Methods Hippocampal subfield volumes were measured in 34 patients with schizophrenia (19 first episode, 6 sub-chronic, 9 chronic) and 15 healthy comparison participants. A subset of 10 first episode and 12 healthy participants were rescanned after six months. Results Total left hippocampal volume was smaller in sub-chronic (p = 0.04, effect size 1.12) and chronic (p = 0.009, effect size 1.42) patients compared with healthy volunteers. The CA2-3 subfield volume of chronic patients was significantly decreased (p = 0.009, effect size 1.42) compared to healthy volunteers. The CA4-DG volume was significantly reduced in all three patient groups compared to healthy group (all p < 0.005). The two affected subfield volumes were inversely correlated with severity of negative symptoms (p < 0.05). There was a small, but statistically significant decline in left CA4-DG volume over the first six months of illness (p = 0.01). Conclusions Imaging strategies defining the subfields of the hippocampus may be informative in linking symptoms and structural abnormalities, and in understanding more about progression during the early phases of illness in schizophrenia.